Prion diseases are a group of fatal neurodegenerative disorders including variant Creutzfeldt-Jakob disease (vCJD) which originates from exposure to bovine spongiform encephalopathy (BSE). Iatrogenic transmission from asymptomatic patients with vCJD prion infection via blood transfusion has had a major impact on public health policy.
Prion Disease encompasses a range of closely related and uniformly fatal neurodegenerative disorders affecting the central nervous system of humans and animals. They include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and kuru in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) of deer and Elk and scrapie in sheep (1;2). The emergence of variant CJD (vCJD), and the confirmation that it originates from exposure to bovine spongiform encephalopathy (BSE) (3;4) has raised a plethora of public health concerns adversely affecting surgery, dentistry, organ transplantation and blood transfusion.
Exposure of the UK population to BSE has been widespread with over 181,000 cases confirmed to date in cattle (5) and yet the prevalence of human infection in the UK has yet to be determined with accuracy. A retrospective study of archived surgical lymphoreticular specimens estimated a prevalence of infection in the UK of 237 per million (95% confidence interval 49-692 per million)(6), far higher than the number of vCJD clinical cases thus far with further studies failing to refine these estimates(7). The demonstration of sub-clinical carrier states of prion infection in animal models (8-10) offers a potential explanation for the discrepancy between clinical cases and supports a prevalence which is likely to be in excess of 1 in 10,000 in the UK (11).
Concern regarding secondary transmission of vCJD has had extensive effects on public health policy in the UK and elsewhere. As the clinically silent incubation period of prion infection in humans can be prolonged, potentially exceeding 50 years (12), and the prevalence is poorly defined, the extent of future transfusion-transmission of vCJD cannot be estimated or accurately risk-assessed. Secondary infection from the clinically silent population has already been confirmed in four recipients of blood transfusion (13-15). Whilst the number of transfusion recipients positively identified as having received vCJD contaminated packed red cells is small, a much larger cohort of around 7000 recipients of contaminated plasma products have already been identified and notified of their at-risk status (16). Concern for this cohort has been heightened by post-mortem evidence for infection with vCJD prions in the spleen of a person with haemophilia (11).
The infectious agents, or prions, responsible for transmission of disease are composed principally if not entirely of a misfolded form of the host prion protein, PrPC. PrPC is ubiquitous, although expressed at highest levels in the central nervous system (CNS) and cells of the immune system. When recruited during prion propagation PrPC is remodelled to an aggregated, detergent insoluble isoform designated PrPSc chemically identical but conformationally distinct from PrPC (17). Detection of PrPSc in CNS and lymphoreticular tissues correlates widely with infection and the presence of prion infectivity (18-20) and is accepted as 100% specific for prion infection.
Although the quantities of PrPSc deposited in neural tissues are sufficient during the symptomatic phase of illness for detection by conventional immunoassays such as western blotting and ELISA, levels in peripheral tissues are significantly lower (21). Quantification of infectious titre using rodent models has indicated that the levels of infectivity, and by inference PrPSc, in blood may be extremely low, with buffy coat fractions containing between 2-10 LD50 Units ml−1 during the asymptomatic phases of disease, rising to 100 LD50 Units ml 1 during the clinical stage (22;23).
In order to successfully identify infection in blood an assay must be able to detect PrP in a range which is several orders of magnitude below the sensitivity of conventionally employed immunoassays. Furthermore, the ratio of background PrPC, which is chemically identical to PrPSc, is higher in blood than any other tissue and the high lipid and protein content of blood also contribute to non-specific background signals. Conventionally, immunoassays for PrPSc have been dependent upon protease pre-treatment of tissues to degrade PrPc and other proteins, thereby reducing cross-reactivity (24). It has now been shown in a number of different studies that the majority of disease associated PrP may well be sensitive to proteolytic digestion with proteinase K (PK) (25-31).
The observation that prions can bind avidly to metal surfaces (32;33) has been used to develop quantitative assays for infectivity (34) that approach the high levels of sensitivity required to detect the low levels of prions and abnormal PrP associated with blood. However, prior art assays typically assess infectivity in an animal model or cell culture system which is labour intensive and time consuming as well as raising ethical issues about use of animals.
Current evidence clearly indicates there is risk of iatrogenic vCJD from transfusion of blood and purified blood components (13-15;36) and by inference from many forms of surgical and dental interventions. Taken together with the knowledge that sub-clinical carrier states of prion disease can exist (8;9) alongside protracted incubation periods for clinical disease which may span several decades (12) the implications for current and future public health are substantial. Current risk reduction strategies in the UK are extremely costly involving leucodepletion of transfused packed red cells and the sourcing of plasma from the USA based on estimates of approximately 40% of infectivity being associated with leucocytes with the remainder in plasma (37). Studies modelling the use of leucocyte depletion have demonstrated reductions in blood infectivity of between 58% (37) and 72% (38). These and related issues also apply outside the UK, for example blood donation is not permitted in the USA if the prospective donor has spent a cumulative period of 6 moths or more in Europe. This causes significant issues for US public and military blood transfusion services.
It remains a major problem that a non-invasive and specific test for prion infection is not available.
The invention seeks to overcome problem(s) associated with the prior art.